Transmitted integrase inhibitor resistance in newly diagnosed HIV remains rare. We report two cases with baseline resistance to all currently available integrase inhibitors in the Netherlands in 2025. Clinical history and laboratory findings indicate possible transmitted resistance. The cases are not phylogenetically linked, representing two independent introductions of integrase inhibitor resistant HIV into the Dutch population. These observations highlight the need for strengthened surveillance and prevention efforts in Europe and globally.

Background Despite effective antiretroviral therapy, HIV-1 remains a global health challenge. Most people living with HIV (PWH) are diagnosed in the chronic stage and around half are diagnosed late globally. Insight into the effect of time of diagnosis and therapy initiation on reservoir dynamics and immune reconstitution within the group with a chronic diagnosis is limited. Methods In this prospective cohort study, PWH diagnosed during the chronic stage (from Fiebig VI) were stratified into late (<350 CD4+ T cells/mm3 or an AIDS-defining illness) or non- late diagnosis subgroups. We analyzed the viral reservoir by IPDA, SQuHIVLa and FISH-Flow, and the immune compartment with the HIV-specific AIM assay and a 45-color spectral flow cytometry panel in the first year after ART initiation. Findings Although proviral DNA decreased during the first year of ART, the inducible reservoir remained stable. One year after ART initiation, exhausted CD8+ T cell abundance correlated significantly with CD4+ T cell count pre-ART. PWH with a late diagnosis had a significantly higher inducible reservoir and lower CD4+ T-cell counts than the non-late HIV diagnosis subgroup. Moreover, the subgroup with a late diagnosis showed higher abundance of exhausted CD8+ T cells, higher expression of activation/exhaustion markers, and lower naive CD4+ T-cell abundance than the non-late diagnosis subgroup. Interpretation Our results show that late diagnosis is associated with a persistently higher inducible viral reservoir and impaired immune recovery. These findings underline the importance of early diagnosis and treatment, and rationalize the use of late diagnosis as a covariate in future cure and reservoir studies.

During dual infection, HIV-1 and HIV-2 may infect the same cell, either simultaneously or sequentially. When these closely related lentiviruses co-infect, HIV-2 has been found to inhibit HIV-1 replication in vitro. In-patient infection data suggests that dual infection attenuates disease progression. Mechanisms underlying this inhibition have not been fully elucidated. Here, we assessed HIV-1 infectivity in presence or absence of HIV-2 in cell culture. HIV-1 subtypes A, B, C, and CRF02_AG were used to infect TZM-bl reporter cells as single infections or as dual infections with HIV-2. Infectious titer concentrations were determined by cytopathic effect-based method (TCID50) and P24 ELISA was used to determine p24 concentration in supernatant. Infectivity was determined using luminescence assay, while qPCR was used to determine expression of cell-associated unspliced and multiply spliced HIV-1 RNA. HIV-1 cell free viral RNA (vRNA) levels were quantified in cell supernatants. Dual infections (simultaneous and sequential) resulted in reduced HIV-1 infectivity and replication relative to single infections. The reduction in infectivity varied among HIV-1 subtypes. Additionally, evaluation of expression levels of unspliced (us) and multiply spliced (ms) HIV-1 RNAs revealed lower expression of these RNA species in simultaneous dual infections for all the four subtypes tested. The same trend was observed for sequential dual infection with the exception of subtype B, which showed a slight increase in levels of usRNA. In both simultaneous and sequential dual infections, expression of msRNA was lower relative to HIV-1 mono-infection. Again, subtype B maintained the same trend as observed in usRNA during sequential dual infection. Despite the observed subtype specific variations, lower HIV-1 infectivity in dual infections coincided with reduced HIV-1 viral loads. In summary, this study demonstrates a significant reduction in HIV-1 infectivity in the presence of HIV-2. Reduced infectivity was characterized by lower viral loads and coincided with reduced HIV-1 transcription and splicing. These findings pave way for future mechanistic studies to understand the drivers of reduced infectivity in dual infection.

The occurrence of virological failure in a subset of individuals is an inevitable aspect of antiretroviral treatment, and historically has been primarily influenced by suboptimal adherence to oral therapies. The risk of selecting 1- or 2-class human immunodeficiency virus (HIV) drug resistance is influenced by the composition of the regimen, differing significantly depending on the intrinsic barrier to resistance of the regimen. HIV resistance emergence during treatment can be viewed as a regimen-related adverse effect that warrants equal consideration in clinical trials alongside virological and safety endpoints. Antiretroviral regimens demonstrating non-inferiority and showing similar rates of virological failure can nonetheless differ in terms of HIV emergent resistance. We propose the development of a systematic framework to categorize emergent HIV drug resistance in clinical trials. Standardizing the evaluation of resistance in clinical trials and its reporting to regulatory agencies will facilitate an improved understanding of regimen-specific resistance risks and better inform clinical decision making.

Objectives Mutations in the 3 '-polypurine tract (3 ' PPT) of HIV-1 have been observed under pressure with two integrase strand transfer inhibitors, dolutegravir and cabotegravir. In the DOMONO randomized clinical trial, 3 ' PPT mutations emerged in a participant who experienced treatment failure under dolutegravir monotherapy. To understand the basis for this rare mutational pathway, we examined baseline viral sequences and identified the K156N natural polymorphism. Given the role of K156 in viral DNA binding, the potential relationship between K156N and 3 ' PPT mutations was further investigated.Methods We assessed the impact of K156N on integrase using in silico modelling and biochemical assays with recombinant proteins. Infectivity, replicative capacity, and drug susceptibility of viruses carrying K156N, 3 ' PPT mutations, or both were measured. Viral evolution was assessed in cell culture.Results Structural models indicated that K156N altered viral DNA binding. K156N reduced strand transfer activity through decreased affinity for the LTR but increased 3 '-processing. The K156N virus had normal infectivity, whereas the 3 ' PPT mutations decreased infectiousness sixfold and lowered maximal infectivity. K156N partially compensated for this defect, but maximal infectivity remained diminished. K156N also partially compensated for defects in replicative capacity imposed by 3 ' PPT mutations. K156N alone did not confer resistance against dolutegravir, nor did it increase the modest (2.5-fold) resistance conferred by the 3 ' PPT mutations. K156N alone promoted the spontaneous emergence of 3 ' PPT mutations distinct from those seen in DOMONO.Conclusions These findings establish a direct functional relationship between natural variation in HIV-1 integrase and the emergence of 3 ' PPT mutations. People harbouring a virus with the K156N natural polymorphism may be predisposed to developing 3 ' PPT mutations upon failure with DTG. However, the clinical relevance of this association remains to be established.

BACKGROUND:Despite effective suppression of HIV-1 replication by antiretroviral therapy (ART), persistent viral reservoirs prevent cure and necessitate lifelong treatment. While short-term dynamics of HIV-1 rebound following treatment interruption have been extensively studied, the longer-term consequences of immune perturbation and transient viraemia for reservoir quality and immune architecture remain incompletely understood. METHODS:We leveraged a rare historical human cohort that experienced prolonged viral rebound during a dendritic cell-based therapeutic vaccine study as a "perturbation model" to explore HIV-1 reservoir characteristics and immune remodelling more than a decade after ART resumption. We combined functional inducible HIV-1 reservoir measurements with high-dimensional immune phenotyping and included comparator analyses using a separate cohort of eight individuals with sustained ART-mediated suppression. FINDINGS:We observed higher inducible HIV-1 reservoir activity in nine participants from the Dendritic Cell-Tat, Rev, and Nef (DC-TRN) trial who previously experienced viral rebound and subsequently achieved viral re-suppression on long-term ART (p = 0.01). This increase in inducible HIV-1 reservoir activity occurred in the absence of major changes in total or intact proviral DNA (p = 0.99, and p = 0.35, respectively). Immune profiling suggested persistent differences in memory T cell phenotypes, after long-term re-suppression on ART, including enrichment of CXCR3-expressing central memory CD4+ T cells that positively correlated with inducible tat/rev msRNA expression (r2 = 0.54, p = 0.03). INTERPRETATION:Together, these findings suggest that immune perturbation and viral rebound may be associated with persistent changes in HIV-1 reservoir inducibility and immune architecture after viral re-suppression. Our study highlights a possible increase in inducible HIV-1 reservoir activity associated with a CXCR3+ memory T cell phenotype, supporting further evaluation of reservoir quality endpoints, candidate immunophenotypic correlates, and long-term monitoring strategies in contemporary remission trials. FUNDING:This research was supported by grants from Aidsfonds (P-60602, P-263, P-53302, P-53601, P-60804), MRACE (EMC2020-083); Horizon Europe (681032); Health Holland (LSHM19100-SGF and EMCLSH19023), and NWO (ZonMW-40-44600-98-333).

INTRODUCTION:HIV remains a major global health concern. Unexpected disruptions in antiretroviral drug supply chains carry increased mortality and transmission risks. The integrase strand transfer inhibitor dolutegravir plays an increasingly critical role in the global fight against the epidemic. Its high barrier to resistance has been extensively documented in rich countries. Recent reports of resistance cases after dolutegravir failure in resource-limited cohorts raise concerns about whether its high barrier to resistance will hold in low-income countries. AREAS COVERED:For this review, we performed a search on the recent published literature and conference communications focused on acquired drug resistance against dolutegravir in low- and middle-income countries. EXPERT OPINION:Overall, the data unsurprisingly showed that resistance against dolutegravir emerged mainly from unsuppressed individuals with treatment adherence issues. This emergence happened at a population rate below 1% despite structural challenges. Almost half of the resistance cases involved the R263K substitution, which did not always preclude re-suppression with dolutegravir. Minor adjustments in the programmatic large-scale rollout of dolutegravir could further improve these outcomes. Continued treatment adherence support and the preservation of antiretroviral drug supply chains remain crucial for the success of HIV treatment.

Cabotegravir plus rilpivirine long-acting (CAB + RPV-LA) injectable therapy marks a major milestone in HIV care, offering an efficacious, well-tolerated alternative to daily oral antiretroviral treatment. This article reviews data from pivotal trials and observational studies of CAB + RPV-LA, addressing emerging questions and highlighting key research priorities. We examine factors influencing virological outcomes, including issues related to HIV subtype, archived drug resistance, body mass index, and pharmacokinetics, and discuss challenges related to hepatitis B virus immunity and infection, pregnancy, and adherence. We outline strategies to address barriers to implementation, advocating for a tailored approach to maximize the potential of CAB + RPV-LA in improving outcomes across diverse populations with HIV.

The presentation of HIV peptides by the human leukocyte antigen (HLA) complex to CD8+ cytotoxic T-cells (CTLs) is critical to limit viral pathogenesis. HIV can mutate to evade HLA-restricted CTL responses and resist antiretroviral drugs, raising questions about how it balances these evolutionary pressures. Here, we used a computational approach to assess how drug resistance-associated mutations (RAMs) affect the binding of HIV-1 subtype B or C peptides to the most prevalent HLA alleles in US, European, and South African populations. We predict RAMs that may be favored in certain populations and report the under-representation of Y181C in people expressing HLA-B*57:01. This finding agreed with our computational predictions when Y181C was at the major anchor site P2, suggesting the potential relevance of our approach. Overall, our findings lay out a conceptual framework to study the implications of HLA alleles on the emergence of HIV RAMs at the individual and population levels.

Background:HIV-2, although less common than HIV-1, exhibits a higher proportion of elite controllers (ECs), who can suppress HIV without antiretroviral therapy (ART), a phenomenon rarely observed in HIV-1. Studying ECs could yield insights into viral control mechanisms and potentially lead to a cure. Methods:We retrospectively characterized a cohort of people with HIV-2 who received care at the Erasmus University Medical Center, Rotterdam, Netherlands. The aim was to identify categories of ECs based on plasma viral loads, CD4+ T-cell count, and responses to ART. Results:Between 1989 and 2023, 52 people with HIV-2 were included, primarily of West African origin (80.8%). Follow-up ranged from <1 to 32 years (median, 16 years). Seven participants were lost to follow-up (13.5.%), and 18 participants died (34.6%), 7 before ART availability due to AIDS. The remaining 40 participants were included in the detailed analysis. Thirteen were ECs with CD4+ T cells >350 cells/mm3 and viral loads <200 copies/mL without use of ART. Four participants progressed to CD4+ T cells <350 cells/mm3 without symptoms of HIV despite undetectable viral loads (nonviremic progressors). Three individuals demonstrated EC status for at least 5 years but lost viral and immunologic control. Nineteen participants exhibited a classical phenotype of viremic progression. Five participants had HIV-1 and HIV-2. Finally, 1 participant had a unique phenotype with loss of control with an unexplained rebound in viremia, followed by resuppression without ART for >10 years (recontroller). Conclusions:These data highlight relevant trajectories among ECs. Understanding the underlying mechanisms can inform decisions on treatment and contribute to finding a cure for all people with HIV.

The occurrence of virological failure in a subset of individuals is an inevitable aspect of antiretroviral treatment, and historically has been primarily influenced by suboptimal adherence to oral therapies. The risk of selecting 1- or 2-class human immunodeficiency virus (HIV) drug resistance is influenced by the composition of the regimen, differing significantly depending on the intrinsic barrier to resistance of the regimen. HIV resistance emergence during treatment can be viewed as a regimen-related adverse effect that warrants equal consideration in clinical trials alongside virological and safety endpoints. Antiretroviral regimens demonstrating non-inferiority and showing similar rates of virological failure can nonetheless differ in terms of HIV emergent resistance. We propose the development of a systematic framework to categorize emergent HIV drug resistance in clinical trials. Standardizing the evaluation of resistance in clinical trials and its reporting to regulatory agencies will facilitate an improved understanding of regimen-specific resistance risks and better inform clinical decision making.

Journal Article Corrected proof Insights in Dolutegravir-Resistance Pathways Get access Thibault Mesplede, Thibault Mesplede Viroscience Department, Erasmus Medical Center, Rotterdam, The Netherlands Search for other works by this author on: Oxford Academic PubMed Google Scholar Pierre Gantner Pierre Gantner Clinical Virology Laboratory, Strasbourg University Hospital, Strasbourg, FranceINSERM UMR_S1109, Strasbourg University, Strasbourg, France Correspondence: P. Gantner, Institut de Virologie, 3 rue Koeberlé, F67000 Strasbourg, France (pierre.gantner@chru-strasbourg.fr). https://orcid.org/0000-0002-3984-883X Search for other works by this author on: Oxford Academic PubMed Google Scholar Clinical Infectious Diseases, ciae127, https://doi.org/10.1093/cid/ciae127 Published: 11 March 2024 Article history Published: 11 March 2024 Corrected and typeset: 30 March 2024

Dolutegravir/lamivudine (DTG/3TC) has a high genetic barrier against the development of human immunodeficiency virus drug resistance. We report 2 cases of R263K + M184V mutations during DTG/3TC failure followed by viral suppression after adherence intervention without treatment change that we attribute to residual drug activity, reduced viral fitness, and robust immune competence.

Background The treatment management of human immunodeficiency virus (HIV)-2 infection presents greater challenges compared to HIV-1 infection, primarily because of inherent resistance against non-nucleoside reverse transcriptase inhibitors. Integrase strand transfer inhibitors, particularly dolutegravir, have improved treatment outcomes for people with HIV-2. Lenacapavir, a novel and potent antiretroviral capsid inhibitor, offers additional therapeutic options. However, limited knowledge exists regarding HIV-2 resistance against dolutegravir and lenacapavir.Methods We report the case of a treatment-experienced individual who did not achieve virological suppression with regimens containing dolutegravir and lenacapavir. Clinical monitoring, genotypic and phenotypic resistance assays, and in silico structural modeling were performed.Results Lenacapavir was added to a failing regimen of boosted darunavir, twice daily dolutegravir, and 2 nucleoside reverse transcriptase inhibitors. Initially, this addition led to a decline in the viral load and increase in CD4+ T-cell count, despite the identification of a previously unreported combination of integrase resistance mutations. However, virological suppression was not achieved and viral load, although reduced, resumed increasing. This rebound was associated with the development of an N73D capsid substitution in HIV-2, which conferred resistance against lenacapavir. Based on cell-based assays predicting hypersusceptibility to bictegravir, the regimen was adjusted to oral lenacapavir plus bictegravir/emtricitabine/tenofovir alafenamide, resulting in a resumption in viral load decline.Conclusions Although lenacapavir demonstrated therapeutic potential, our case underscores the critical need to combine it with other fully active antiretroviral agents to prevent the rapid emergence of resistance and achieve long-term virological control in treatment-experienced individuals with HIV-2. HIV-2 treatment options are limited, and resistance mechanisms to integrase and capsid inhibitors are understudied. Despite emerging resistance, lenacapavir shows promise in salvage therapy, optimally when combined with drugs that have high barriers to resistance like dolutegravir or bictegravir.

The recently Food and Drug Administration (FDA)-approved cabotegravir (CAB) has demonstrated efficacy as an antiretroviral agent for HIV treatment and prevention, becoming an important tool to stop the epidemic in the United States of America (USA). However, the effectiveness of CAB can be compromised by the presence of specific integrase natural polymorphisms, including T97A, L74M, M50I, S119P, and E157Q, particularly when coupled with the primary drug-resistance mutations G140S and Q148H. CAB's recent approval as a pre-exposure prophylaxis (PrEP) may increase the number of individuals taking CAB, which, at the same time, could increase the number of epidemiological implications. In this context, where resistance mutations, natural polymorphisms, and the lack of drug-susceptibility studies prevail, it becomes imperative to comprehensively investigate concerns related to the use of CAB. We used molecular and cell-based assays to assess the impact of T218I and T218S in the context of major resistance mutations G140S/Q148H on infectivity, integration, and resistance to CAB. Our findings revealed that T218I and T218S, either individually or in combination with G140S/Q148H, did not significantly affect infectivity, integration, or resistance to CAB. Notably, these polymorphisms also exhibited neutrality concerning other widely used integrase inhibitors, namely raltegravir, elvitegravir, and dolutegravir. Thus, our study suggests that the T218I and T218S natural polymorphisms are unlikely to undermine the effectiveness of CAB as a treatment and PrEP strategy.

Human immunodeficiency virus (HIV) treatment with antiretroviral regimens containing integrase strand transfer inhibitors such as dolutegravir (DTG) and bictegravir (BIC) offers high levels of protection against the development of drug resistance mutations. Despite this, resistance to DTG and BIC can occur through the development of the R263K integrase substitution. Failure with DTG has also been associated with the emergence of the G118R substitution. G118R and R263K are usually found separately but have been reported together in highly treatment-experienced persons who experienced treatment failure with DTG. We used cell-free strand transfer and DNA binding assays and cell-based infectivity, replicative capacity, and resistance assays to characterize the G118R plus R263K combination of integrase mutations. R263K reduced DTG and BIC susceptibility ~2-fold, in agreement with our previous work. Single-cycle infectivity assays showed that G118R and G118R plus R263K conferred ~10-fold resistance to DTG. G118R alone conferred low levels of resistance to BIC (3.9-fold). However, the G118R plus R263K combination conferred high levels of resistance to BIC (33.7-fold), likely precluding the use of BIC after DTG failure with the G118R plus R263K combination. DNA binding, viral infectivity, and replicative capacity of the double mutant were further impaired, compared to single mutants. We propose that impaired fitness helps to explain the scarcity of the G118R plus R263K combination of integrase substitutions in clinical settings and that immunodeficiency likely contributes to its development.

Analytical treatment interruption (ATI) studies are increasingly being performed to evaluate the efficacy of putative strategies towards HIV-1 reservoir elimination or antiretroviral therapy (ART)-free viral control. A limited number of studies have evaluated the impact of ATI on the HIV-1 reservoir in individuals on suppressive ART. Available data suggests that ATIs have transient impact on the HIV-1 reservoir, mostly measured by levels of total or integrated HIV-1 DNA, in peripheral blood cells prior to ATI and shortly after ART-mediated viral re-suppression. The long-term impact of intervention ATI studies on the latent, inducible HIV-1 reservoir remains uncertain. We report the first clinical study demonstrating an increase in the latent, inducible HIV-1 reservoir, measured by expression of tat/rev multiply spliced RNA, in nine individuals, despite more than a decade of re-suppressive ART, after undergoing an immune intervention ATI conducted in 2006-2009. Our findings challenge the status quo on ATI risk of viral reservoir reseeding and the long-term outcomes thereof. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement C.L. received funding from the Dutch Aidsfonds (grants P-60602 and P-263); SR received funding from Dutch Aidsfonds (grant P-53302); C.R. received funding from Dutch Aidsfonds (grant P-53601); R.A.G. received funding from Dutch Aidsfonds (grant P-60804) and Horizon Europe (grant 681032);TMa received funding from Health Holland (grants LSHM19100-SGF and EMCLSH19023) and ZonMW (grant 40-44600-98-333) and a Building Synergistic Infrastructure for NL4Cure grant. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Erasmus University Medical Center, Rotterdam gave ethical approval for this work (MEC-2005-227 and MEC-2012-583). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data needed to evaluate the results and conclusions in this article are provided in the main text, Methods section and Extended data section. Additional data related to the study may be requested from the lead contact T. Mahmoudi. Unique biological material, where available, can be obtained upon request.

Reactivation of the latent HIV-1 reservoir is a first step toward triggering reservoir decay. Here, we investigated the impact of the BAF complex inhibitor pyrimethamine on the reservoir of people living with HIV-1 (PLWH). Twenty-eight PLWH on suppressive antiretroviral therapy were randomized (1:1:1:1 ratio) to receive pyrimethamine, valproic acid, both, or no intervention for 14 days. The primary end point was change in cell-associated unspliced (CA US) HIV-1 RNA at days 0 and 14. We observed a rapid, modest, and significant increase in (CA US) HIV-1 RNA in response to pyrimethamine exposure, which persisted throughout treatment and follow-up. Valproic acid treatment alone did not increase (CA US) HIV-1 RNA or augment the effect of pyrimethamine. Pyrimethamine treatment did not result in a reduction in the size of the inducible reservoir. These data demonstrate that the licensed drug pyrimethamine can be repurposed as a BAF complex inhibitor to reverse HIV-1 latency in vivo in PLWH, substantiating its potential advancement in clinical studies.